Kinetic Reaction Mechanism of Sinapic Acid Scavenging NO2 and OH Radicals: A Theoretical Study.

The mechanism and kinetics underlying reactions between the naturally-occurring antioxidant sinapic acid (SA) and the very damaging ·NO2 and ·OH were investigated through the density functional theory (DFT). Two most possible reaction mechanisms were studied: hydrogen atom transfer (HAT) and radical adduct formation (RAF). Different reaction channels of neutral and anionic sinapic acid (SA-) scavenging radicals in both atmosphere and water medium were traced independently, and the thermodynamic and kinetic parameters were calculated. We find the most active site of SA/SA- scavenging ·NO2 and ·OH is the -OH group in benzene ring by HAT mechanism, while the RAF mechanism for SA/SA- scavenging ·NO2 seems thermodynamically unfavorable. In water phase, at 298 K, the total rate constants of SA eliminating ·NO2 and ·OH are 1.30×108 and 9.20×109 M-1 S-1 respectively, indicating that sinapic acid is an efficient scavenger for both ·NO2 and ·OH.

Introduction

Sinapic acid (SA, 3,5-dimethoxy-4-hydroxycinnamic acid) is a naturally-occurring and widespread phenolic acid in the plant kingdom, and can be obtained from various fruits and vegetables such as rye [1], orange, grapefruit, and cranberry [2]. Especially, SA accounts for over 73% of all free phenolic acids in rapeseed [3]. SA is a bio-active compound reported as anti-inflammatory and anxiolytic ingredient [4,5]. In addition, SA is a widely-investigated antioxidant, and because of the peroxynitrite (ONOO-) scavenging activity [6], it can be utilized to promote the cellular defense activity against the diseases involving ONOO- [7].

Hydroxycinnamic acid is a particularly important group of phenolic acids, mainly including caffeic acid, ferulic acid, p-coumaric acid, and sinapic acid, which exist in daily foods such as grape, citrus [8], pear, tomato, and small radish [2]. Antioxidant activities of hydroxycinnamic acids have been studied extensively [9-12]. Among, SA is suggested as a more efficient antioxidant [13] and superoxide radical scavenger [14] than p-coumaric acid and ferulic acid. Cuvelier et al. have also confirmed that, in a lipophilic solvent, the antioxidant activity of SA is higher than that of p-coumaric acid, ferulic acid, and syringic acid [15].

Owing to the high radical scavenging ability, SA is a good protector against the oxidative damage caused by the accumulation of redundant free radicals in vivo, which are the primary cause of many chronic diseases [16-20]. Thus, it is vital to analyze the viable mechanisms underlying how SA eliminates different radicals. Despite having some experimental research, there are only three theoretical studies about the antioxidant capacity of SA to our best knowledge. First, Bakalbassis et al. investigated the structure-antioxidation relationships of SA and other three hydroxycinnamic acid derivatives using ab initio and DFT methods. By calculating the absolute infrared intensity, heat of formation, and electron-donating ability, they evaluated the antioxidant activities of SA and other derivatives, and found that both degree of conjugation and extent of spin delocalization in phenoxyl radicals affect the scavenging activity of phenolic acid [21]. Second, Galano et al. theoretically studied the ·OOH scavenging activity of SA in aqueous and lipid solutions using DFT. They predicted the rate coefficient of SA with ·OOH, and concluded SA is a good ·OOH scavenger [22]. Third, Urbaniak et al. studied the antioxidant properties of p-coumaric acid and SA, and obtained the bond dissociation enthalpy, adiabatic ionization potential, proton dissociation enthalpy, proton affinity, electron transfer enthalpy, gas phase acidity, and Gibbs energy at the DFT/B3LYP. They confirmed the high antioxidant activities of both compounds, and indicated O-H is the preferred place of free radical attack [23].

It can be seen the theoretical research concerning the antioxidant activity of SA are really little. Even for the theoretical research of phenolic acids in the same area, the focus are almost on the structural and electronic properties of antioxidants, such as bond dissociation energy (BDE), adiabatic ionization potential (AIP), and spin density (SD) etc [23-30]. The reaction mechanisms of phenolic acids with specific radicals are rarely investigated. Moreover, reactive oxygen species (ROS) attracted more attention [21–22, 31–33], while another main byproducts in human body: reactive nitrogen species (RNS) are unnoticed. Thus, the main goal of this work is to carry out a systematical theory study on the reaction mechanisms of SA with two important RNS and ROS (·NO2 and ·OH), and provide the thermodynamic and kinetic details of all possible reaction channels. Hope our results are helpful for designing the high-activity scavengers against RNS and ROS.

·NO2 and ·OH were chosen because they are the typical radicals of RNS and ROS, respectively, in biosystems. Excessive production of RNS and ROS may exert pathological stress to cells and tissues. This oxidative stress can weaken the defense system and render the tissues more sensitive to the subsequent insult. Thus, such mechanism can cause cell damages including oxidative damage to essential proteins, lipid peroxidantion, and DNA strand breakage. As a common RNS, ·NO2 reportedly can induce lipid peroxidation, membrane damage and cell death [34-36]. And in aqueous solution, ·NO2 could be hydrolyzed to ONOO-, NOO- or other like harmful species, so it is significant to investigate the ·NO2 eliminating mechanism. As for ·OH, it is the most active and damaging ROS in biosystems, with a very short half-life of about 10−9 s [37]. Thus, the study of kinetic mechanisms how SA scavenges these two radicals are valuable to the further design of phenolic antioxidants.

It is generally assumed that the reactions between phenolic compounds and radicals (·R) proceeds through three main mechanisms: direct H-transfer process from the antioxidant molecule (Eq 1), radical adduct formation (Eq 2) and single-electron transfer process (Eqs 3 and 4):

Hydrogen atom transfer (HAT):

Radical adduct formation (RAF):

Single-electron transfer (SET):

The previous theoretical studies on the homologous phenolic compounds such as gallic acid, caffeic acid, and sinapic acid have shown that SET mechanism dose not play a relevant role in the reactions with free radicals [31, 38–40]. Hence, in the present work, we are focused on HAT and RAF as the most probable reaction mechanisms.

The contribution of two mechanisms were analyzed. All possible reaction/attack sites were examined, and the corresponding channels were identified by thermodynamic and kinetic calculations. Rate constants and branching ratios for different channels were also estimated. To simulate the water-dominated cellular environment, we also took into account the effect of aqueous solution. Then the theoretical and experimental results were compared.

Computational Methods

DFT calculations were carried out using the GAUSSIAN 09 computational package [41]. Geometry optimization and frequency calculations were performed at the M05-2X level [42] with the basis set 6–311++G(d,p). The M05-2X functional was recommended for kinetic calculation by the developers [43] and was successfully used by independent authors with that purpose [44-51]. It is also one of the best functional for calculating the reaction energy involving free radicals [52].

Unrestricted calculations were used for open shell systems. The nature of stationary points was evaluated by using normal vibration frequencies: all of reactants (R), complexes (C) and products (P) must show positive real frequencies; all of transition states (TSs) must show a single imaginary frequency which corresponds to the expected vibration mode. In addition, the intrinsic reaction coordinates (IRC) at the M05-2X/6-311++G(d,p) level were calculated to obtain the minimum energy path.

Solvent effects were introduced using the continuum solvation model based on solute electron density (SMD) [53], which is a universal solvation model due to its applicability to either charged or uncharged solute in any solvent or liquid medium. At the same level of M05-2X/6-311++G(d,p), all reactants and products were optimized in aqueous solution by SMD model. The transition states were also optimized using SMD model in aqueous solution. However, in spite of trying our best, only part of the TS for water-phase pathways were identified. Therefore, the solvation effect on TS were estimation by the single point calculations with SMD model on the basis of the optimized gas-phase geometries. Then, we compared the geometries of TS found in aqueous solution with the corresponding TS optimized in gas phase (see S1 Fig). The results show that the geometries of TS optimized in aqueous solution are very similar with that of TS optimized in the gas phase. Such as, for the bond of readying to form, the biggest difference in bond length is only 0.13Å. We also compared the energy barrier heights obtained by optimization of TS under SMD model with that of obtained by the single point calculation using SMD model based on the gas-phase optimized geometries of TS (see S1 Table). It can be seen that the differences in energy barrier height are also little. Hence, we think that the single energy calculation using SMD model seems to provide a proper estimation of solvent effects for our studied systems. Just as reported in the literature [54-56], the results of single point calculations using solvent model were in good agreements with the corresponding experimental results for their systems.

Reaction enthalpy in solution was computed by the difference of enthalpy values between products and reactants optimized in the presence of SMD model. Relative Gibbs energy in solutions was computed using thermodynamic cycle and Hess’ law which explicitly include solvation energy. For example, the thermodynamic cycle for the addition reaction between SA and ·OH is as follows: With this strategy, the Gibbs energy of reaction in solution (ΔGsol) can be determined as the sum of the Gibbs energy of reaction in the gas phase (ΔGgas) and the difference in solvation energies (ΔΔGs): where ΔΔGs is calculated as: where ΔGs is the solvation energy. The reference state is 1M in all cases. The solvent cage effect was included with Okuno’s corrections [57], which take into account the free volume theory. These corrections agree well with those independently obtained by Ardura et al. [58]. In this work the expression used to correct the Gibbs energy as follows: where n is the reaction molecularity. According to Eq 8, the solvent cage effect causes a decrease of 10.63 kJ/mol in ΔG for a bi-molecular reaction at 298.15 K [59].

The theoretical rate constants of all channels were calculated using the theory of improved canonical variational transition state (ICVT) [60] with small-curvature tunneling (SCT) correction [61] on the program POLYRATE 9.7 [62]. We adopted a separable equilibrium solvation (SES) approximation [63] to calculate the rate constants of water-phase reactions. Specifically, we first calculated the gas-phase reaction channels, solved each configuration along the gas-phase IRC (including reactants, products and saddle points), and then calculated the water-phase rate constants through the variational transition state theory with interpolated single-point energy (VTST-ISPE) on POLYRATE.

Results and Discussion

The optimized structure of SA as well as the atomic numbering scheme are shown in Fig 1. Clearly, SA has an approximately planar structure in which the dihedral angle between benzene ring and carbonyl group is about 179.56°. This result is consistent with the reported structure of SA optimized under the B3LYP/6-31+G(d) level [64]. The planar structure of SA implies that the molecule is completely conjugated and lead to an extended spin delocalization [65]. The possibility of excellent delocalization could account for its potential radical scavenging activity [65].

Fig 1

The optimized geometry of SA in the gas phase.

The result show that SA has an approximately planar structure. The dihedral angle between benzene ring and carbonyl group is ~179.56.

·NO2 scavenging by SA

For reactions of SA with ·NO2, we considered two most possible HAT channels from -OH and -COOH, and defined them as channels O15 and O22, respectively (on sites O15 and O22 in Fig 1); we also considered two RAF channels with addition of the corresponding radical to the carbon-carbon double bond, and defined them as channels C11 and C12, respectively (on sites C11 and C12 in Fig 1).

The optimized geometries of TSs and product complexes (PCs) of all channels are shown in Figs 2 and 3. The reaction enthalpy (ΔH), ΔG and energy barrier height (ΔE) including zero-point energy (ZPE) corrections obtained for each channel were collected in Table 1.

Fig 2

The transition state geometries for the reactions of SA with ·NO2.

Channel O15 has an earlier TS relative to channel O22, associated with a lower reaction energy barrier. This agree with our calculation results in Table 1.

Fig 3

The optimized geometries of product complexes for the reactions of SA with ·NO2.

In HAT product complexes structures, HONO molecules form hydrogen bonds with SA semiquinone radicals.

Table 1

The reaction enthalpies (ΔH), reaction Gibbs energies (ΔG) and energy barrier heights with ZPE corrections (ΔE+ZPE), at 298 K, for the reactions of SA with ·NO2 in the gas phase and water phase (in kJ/mol).

SA+·NO2	ΔH gas	ΔH sol	ΔG gas	ΔG sol	ΔE gas+ZPE	ΔE sol+ZPE
O15	19.09	-7.63	16.78	-25.42	42.58	-8.79
O22	142.85	57.59	136.15	-58.39	109.25	117.18
C11	26.76	11.70	77.34	51.90	57.39	44.84
C12	-0.63	-12.57	51.82	29.61	50.29	32.15

According to the values of ΔG (Table 1), only HAT channels in water phase are exergonic (ΔG<0), meaning they could spontaneously occur. On the contrary, the RAF channels are all endergonic (ΔG>0) wherever in gas or water phase. Such, we conclude that the RAF mechanism is not thermodynamically feasible for SA scavenging ·NO2.

As showed in Fig 2, within the HAT reactions, the TS O15 appears systematically earlier than the TS O22. Since the earlier TS is usually associated with lower reaction energy barrier, the site O15 should be more active. The results of ΔE+ZPE (Table 1) indeed show such a trend: channel O15 has lower energy barrier height than channel O22 both in gas and water phases. For RAF reactions, channel C11 and C12 have similar energy barrier heights and thus similar activities. The active order in terms of energy barrier height is as follows: O15 (42.58 kJ/mol) > C11 (50.29 kJ/mol) > C12 (57.39 kJ/mol) > O22 (109.25 kJ/mol) in gas phase, and O15 (-8.79 kJ/mol) > C11 (32.15 kJ/mol) > C12 (44.84 kJ/mol) >O22 (117.18 kJ/mol) in water phase. Thus, the medium does not seem to affect the order of reaction energy barrier or activity. In this case, channel O15 is the major channel for all reactions of SA with ·NO2. In addition, the water-phase channel O15 with negative ΔE+ZPE is a barrierless reaction, meaning a pre-reactive complex (IM) is formed in the entrance of the reaction, which energy is lower than that of the reactants, and the “real” energy barrier height between IM O15 and TS O15 is 4.57 kJ/mol. The details of relative energies are plotted in Figs 4 and 5.

Fig 4

The potential energy surfaces for the reactions of SA with ·NO2 in the gas phase.

The relative energies (in kJ/mol) were calculated at the M05-2X/6-311++G(d,p) + ZPE level. To facilitate the comparison, the energy of the reactants are set to zero.

Fig 5

The potential energy surfaces for the reactions of SA with ·NO2 in water phase.

The relative energies (in kJ/mol) were calculated at the M05-2X/6-311++G(d,p) + ZPE level. To facilitate the comparison, the energy of the reactants are set to zero.

The spin density is another important parameter to characterize the stability of free radicals formed from phenolic acids, because the energy of a free radical can be efficiently decreased if the unpaired electron is highly delocalized through the conjugated system [66]. The phenolic acid radical having higher spin density delocalization, easier is the formation of product and then higher radical-scavenging activity of the group. Atomic spin densities plots for the product radicals of all water-phase channels in S3 Fig indicate that when H-abstraction occurs on O15 atom, a broad delocalization of the unpaired electron involving the O15, almost all C atoms of benzene ring, C11 and C12 atoms occurs. While the RAF channels entail a small delocalization of the odd electron concentrating on the carbon-carbon double bond. The spin density of O15 atom (0.243) of Product O15 shows lager spin density concentration than O22 atom (0.016) of Product O22, which is further confirmed that O15H-group is more sensitive to the free-radical attack.

The rate constant and branching ratio of each channel, as well as the total rate constants at 298 K in both gas and water phases, were calculated out and listed in Table 2. We assumed that neither mixing nor crossover between different channels occurs and therefore calculated the total rate constants of HAT and RAF reactions for SA scavenging ·NO2 as follows:

Table 2

The calculated ICVT/SCT rate constant (k) and branching ratios (Γ) for SA scavenging ·NO2, at 298 K, in the gas phase and water phase, together with the available experimental value from [67].

SA+·NO2	atmosphere		water		experiment
	k (M-1 S-1)	Γ(%)	k (M-1 S-1)	Γ(%)	k (M-1 S-1)
O15	3.06X105	98.92	1.28X108	99.99	7.2X108
O22	3.18X103	1.03	3.58X103	0.01
C11	4.94X101	0.02	9.51X101	~0
C12	1.02X102	0.03	1.58X102	~0
Total	3.1X105		1.3X108

The rate constant of each mechanism was estimated by summing up the rate constants of different channels:

As showed in Table 2, channel O15 possesses the largest rate constant which is several orders of magnitude higher than other channels. The RAF channels C11 and C12 have similar rate constants, but which are much lower than HAT channels. In addition, for the same channel, the k in water phase is always higher than in gas phase, which is particularly evident on the channel O15, indicating that aqueous solutions can enhance the ·NO2 scavenging activity of SA.

To quantify the contribution of each channel to the total reaction, we calculated the branching ratio (Γ), representing the percent of each channel in the total rate constant, as follows:

Also in Table 2, channel O15 contributes more than 98% capacity of scavenging ·NO2, indicating it accounts for almost the whole activity of SA. And the rest channels are very rarely used. Summarily, channel O15, combined with its thermodynamic superiority, is the absolutely dominant pathway for SA scavenging ·NO2. As for the RAF channels, since their total branching ratio is very little and they are significantly endergonic, it is reasonable that the RAF mechanism is irrelevant for the ·NO2 scavenging activity of SA in gas or water phase.

To better understand the kinetic mechanisms of the studied reactions, we calculated the temperature dependence of rate constant for each channel and plotted it against the reciprocal of temperature in Figs 6 and 7. Clearly, the HAT channel O15 is always keeping its superiority in the range of 200–600 K, which further confirms its leading role. The lower lines kO22, kC11 and kC12 are nearly overlapped, especially at lower temperature. The rate constant of channel O15 presents a negative temperature dependence, as its negative energy barrier (Fig 7).

Fig 6

The calculated ICVT/SCT rate constants for the reactions of SA with ·NO2 and the total rate constants (ktotal-NO2) versus 1,000/T, from 200 K to 600 K, in the gas phase (in M-1 S-1).

All of HAT and RAF channels have positive temperature dependence.

Fig 7

The calculated ICVT/SCT rate constants for the reactions of SA with ·NO2 and the total rate constants (ktotal-NO2) versus 1,000/T, from 200 K to 600 K, in water phase (in M-1 S-1).

The HAT channel O15 has a negative temperature dependence, as a negative energy barrier within the reaction process; other channels have positive temperature dependence.

The total rate constants at 298 K, as well as the experimentally- determined rate constant [67], are also listed in Table 2. In aqueous environment, the calculated total rate constant of SA scavenging ·NO2 is 1.30×108 M-1 S-1, which is at the same order of magnitude as the experimental result (7.20×108 M-1 S-1). This agreement validates the reliability of our calculations. Furthermore, it is important to compare the rate constant of SA scavenging ·NO2 with the rate constants of ·NO2 damaging unsaturated fatty acids in vivo. As reported, the rate constants in ·NO2-based oxidations of tyrosine, fumaric acid and linoleic acid are (M-1 S-1): k = 3.20×105 [68], 1.30×107 and 5.00×104 [69], respectively, which are much smaller than the rate constant of SA eliminating ·NO2 predicted here. Thus, we conclude that SA is able to efficiently prevent cell damage by directly trapping ·NO2.

·OH scavenging by SA

Like the previous section, in order to investigate the antioxidant activity of SA toward ·OH, we identified two HAT channels O15h and O22h, as well as two RAF channels C11h and C12h. Because we concern the application of the antioxidant in vivo, and the obtained rate constants in aqueous solutions are closer to the fact in vivo environment, we only retained the thermodynamic and kinetic data of water phase in this section.

The results of ΔH, ΔG and ΔE including the ZPE corrections calculated under the level of M05-2X/6-311++g(d,p) are listed in Table 3. For scavenging ·OH, all HAT and RAF channels in aqueous solutions are exothermic and exergonic, indicating they are all thermodynamically feasible. Thus, SA is able to scavenge ·OH in vivo through both mechanisms of HAT and RAF. According to the spin densities plots in S3 Fig, similar with scavenging ·NO2, HAT channels have more extended delocalization of the unpaired electron, indicating that the HAT mechanism should be more efficient than the RAF mechanism.

Table 3

The reaction enthalpies (ΔH), reaction Gibbs energies (ΔG) and energy barrier heights with ZPE corrections (ΔE+ZPE), at 298 K, for the reactions of SA with ·OH in water phase (in kJ/mol).

SA+·OH	ΔH sol	ΔG sol	ΔE sol+ZPE
O15h	-169.11	-183.65	-54.14
O22h	-103.89	-216.61	-21.85
C11h	-126.86	-95.06	-3.20
C12h	-138.87	-104.48	-3.39

As showed in Table 3, channel O15h has a lower energy barrier height than channel O22h, it is the major channel of SA scavenging ·OH within the HAT mechanism. The -O15H group has higher activity mainly because the corresponding H-abstraction product is a more stable semiquinone radical, as the spin density of O15h atom (0.243) of Product O15h has lager spin density concentration than O22h atom (0.016) of Product O22h. The activities of RAF channels are lower than HAT channels, and channels C11h and C12h are very little different in energy barrier heights. As mentioned before, the channels with negative energy barrier heights are barrierless reactions, and the IMs are formed in the entrances of the reactions. The “real” energy barrier heights need to overcome between IM O15h and TS O15h, IM O22h and TS O22h, IM C11h and TS C11h, IM C12h and TS C12h are 58.71, 30.74, 7.49 and 1.90 kJ/mol, respectively. The relative energies are plotted in Fig 8.

Fig 8

The potential energy surfaces for the reactions of SA with ·OH in water phase.

The relative energies (in kJ/mol) were calculated at the M05-2X/6-311++G(d,p) + ZPE level. To facilitate the comparison, the energy of the reactants are set to zero.

The rate constants and branching ratios of SA scavenging ·OH were also calculated and listed in Table 4, as well as the available experimental data. As showed in Table 4, the HAT channel O15h possesses the largest rate constant, followed by the HAT channel O22h. The RAF channels C11h and C12h have similarly lower rate constants. Thus, to the total reaction of SA with ·OH, the most contributive pathway is channel O15h (ΓO15h = 62.97%). And different from the case of scavenging ·NO2 (ΓO22 = 0.01%), channel O22h has a considerable contribution (ΓO22h = 36.98%). The contributions of RAF channels (ΓRAF = 0.05%) are estimated to be negligible. The total rate constants of HAT channels are on the 109 at 298 K, meaning that the reactions are very fast and diffusion controlled, underlining the excellent antioxidant activity of SA toward ·OH.

Table 4

The calculated ICVT/SCT rate constant (k) and branching ratios (Γ) for SA scavenging ·OH, at 298 K, in water phase, together with the available experimental values (kexp) from [70].

site	k (M-1 S-1)	Γ(%)	kexp (M-1 S-1)
O15h	5.79X109	62.97	9.6X109
O22h	3.40X109	36.98
C11h	3.46X106	0.03
C12h	2.30X106	0.02
Total	9.2X109

The temperature dependence of rate constants against the reciprocal of temperature for each channel and total reaction are sketched in Fig 9. It is clear that channel O15h always maintains the largest rate constant from 200–600 K. The second channel O22h is also very important, and its ratio branching is increasingly higher with the temperature rising. In addition, the rate constants of HAT channels have negative temperature dependence, while the RAF channels have positive temperature dependence.

Fig 9

The calculated ICVT/SCT rate constants for the reactions of SA with ·OH and the total rate constants (ktotal-OH) versus 1,000/T, from 200 K to 600 K, in water phase (in M-1 S-1).

The HAT channels O15h and O22h have negative temperature dependence, the RAF channels C11h and C12h have positive temperature dependence.

According to the above points of view, thermodynamics and kinetics: among the two feasible mechanisms, HAT is the major mechanism for the antioxidant activity of SA toward ·OH in vivo. Channel O15h is the major pathway of all reactions, followed by channel O22h, which is also not negligible. Although the RAF mechanism is thermodynamically feasible, its contribution to the total reaction is negligible.

The theoretical total rate constant at 298 K computed here (9.20×109 M-1 S-1) agrees well with the pulse radiolysis experiment about the ·OH-scavenging capability of SA conducted in neutral solution (k = 9.60×109 M-1 S-1) [70], which further supports the reliability of the calculations in our work.

In aqueous solutions, the ·OH-scavenging activity of SA was higher than other hydroxycinnamic acids, including ferulic acid (4.55×109 M-1 S-1) [71], caffeic acid (3.24×109 M-1 S-1) [72] and gallic acid (7.02×108 M-1 S-1) [21]. Therefore, SA is an excellent ·OH scavenger among the hydroxycinnamic acid antioxidants.

Radicals scavenging by SA-

In the environment of physiological pH (7.4), SA primarily exist in anionic form with a dissociated COOH group [73-74]. In order to provide a detailed investigation on the radical scavenging activity of SA toward ·NO2 and ·OH, also the anionic form of sinapic acid has been taken into account, considering HAT and RAF mechanisms in aqueous.

For each radical, one HAT channel (O15 for ·NO2; O15h for ·OH) and two RAF channels (C11, C12 for ·NO2; C11h, C12h for ·OH) were considered. Under the M05-2X/6-311++G(d,p) level, SA- also has a planar stricture, the optimized geometries of SA-, TSs and PCs of all channels are gathered in S5 Fig. The ΔH, ΔG and ΔE+ZPE of all water-phase channels were also calculated and listed in Table 5.

Table 5

The reaction enthalpies (ΔH), reaction Gibbs energies (ΔG) and energy barrier heights with ZPE corrections (ΔE+ZPE), at 298 K, for the reactions of SA- with ·NO2 and ·OH in water phase (in kJ/mol).

SA-+·NO2	ΔH sol	ΔG sol	ΔE sol+ZPE	SA-+·OH	ΔH sol	ΔG sol	ΔE sol+ZPE
O15	-14.73	-34.77	19.35	O15h	-176.21	-192.99	-10.12
C11	6.37	62.16	35.89	C11h	-161.61	-96.31	-8.73
C12	-36.34	10.43	15.98	C12h	-134.18	-113.51	-8.03

For scavenging ·NO2, according to the Table 5, the channel C12 has the lowest energy barrier height, but the ΔG of this channel is positive, meaning that it is could not occur spontaneously. While the channel O15, with a second lower energy barrier height, is the largest exergonic channel, hence it is the major channel of SA with ·NO2. The ΔG values of 62.16 and 10.43 kJ/mol for the RAF channels C11 and C12 indicate that this mechanism is quite unfavored also when the antioxidant is present in the anionic form, and thus it can be concluded that both SA and SA- do not follow the RAF process when scavenging ·NO2 radicals.

For scavenging ·OH, all channels are exothermic and exoergic, indicating both HAT and RAF mechanisms are feasible. Among them, the channel O15h has a absolute dominance, as it is the most exothermic and exoergic channel, and having the lowest energy barrier height. These results are in agreement with the scavenging activity of neutral SA toward ·OH.

Considering the largest contribution channel O15/O15h, the energy barrier heights of SA- scavenging ·NO2 and ·OH are much higher than those of SA, thus the rate constants of SA- scavenging both radicals should be lower than the rate constants of SA scavenging ·NO2 and ·OH.

Conclusions

In this work, we carried out a systematic study on the radical scavenging activities of SA and SA- toward ·NO2 and ·OH in aqueous simulated media using DFT and direct dynamic method.

For SA/SA- scavenging,·NO2, only HAT reactions in water phase are thermodynamically feasible. The H-abstraction reaction from site O15 is the major channel of all reactions, while the RAF mechanism is not useful.

For SA/SA- scavenging ·OH, both HAT and RAF mechanisms are very feasible thermodynamically and kinetically. In the HAT mechanism, the most active site is also the -O15H group in benzene ring. The RAF mechanism is weaker than HAT, and the activities of two channels (C11h and C12h) are similar.

The reactions of SA eliminating ·NO2 and ·OH take place predominantly by the HAT mechanism (Γ>99%), especially via the channel O15/O15h. For the reactions of SA with ·OH, the site O22h on the carboxyl group is also a significantly active site. Agreeing with our points, the DFT study from Galano et al. reported that the main mechanism of SA scavenging ·OOH should be HAT with the largest contribution (Γ≈99.9%), and the most important active site is the phenolic group of SA to scavenge ·OOH [22].

The total rate constants of SA scavenging ·NO2 and ·OH are 1.30×108 and 9.20×109 M-1 S-1 respectively, in water phase, at 298 K; and are 1.10×108 and 8.20×109 M-1 S-1 respectively, at 310 K. The rate constants in pulse radiolytic experiments are 7.20×108 and 9.60×109 M-1 S-1, respectively. The total rate constant of ktotal-NO2 is small than ktotal-OH, due to the high activity of ·OH, which is a reasonable result, but they are still very fast. Thus we state SA can efficiently scavenge ·NO2 and ·OH radicals in vivo.

The comparison of geometries between the TS optimized under SMD model and the TS optimized in the gas-phase.

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The transition state geometries for the reactions of SA with ·OH.

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The spin density plots for the products of SA scavenging ·NO2 and ·OH.

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The optimized geometries of reactants and products in aqueous solution for the reactions of SA with ·NO2 and ·OH.

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The optimized geometries of SA-, TSs and PCs for the reactions of SA- with ·NO2 and ·OH.

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The energy barrier heights obtained by optimization of TS under SMD model and the energy barrier heights obtained by the single point calculation with SMD model based on the gas-phase optimized geometries of TS.

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The geometry coordinates of all species optimized at M05-2X/6-311++G(d,p) level.

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